Malaria is present in more than one hundred countries and according to the World Health Organization, there were an estimated 198 million cases of malaria worldwide (range 124-283 million) in 2013, and an estimated 584 000 deaths (range 367 000-755 000). 90% of all malaria deaths occur in Africa.
Malaria is caused by Plasmodium parasites. These parasites are spread to people through the bites of infected Anopheles mosquitoes, called “malaria vectors”, which bite mainly between dusk and dawn. There are four protozoa parasite species that cause malaria in humans: Plasmodium (P.) falciparum; P. vivax; P. malariae and P. ovale. The parasites P. falciparum and P. vivax are the most common. Death by malaria is almost exclusively caused by P. falciparum. 
Most of the drugs currently used for treating malaria patients include chloriquine, atovaquone-proguanil (Malarone®), artemether-lumefantrine (Coartem®), mefloquine (Lariam®), quinine, quinidine, doxycycline (used in combination with quinine), clindamycin (used in combination with quinine), artesunate.
Presently, there is a growing need for antimalarial agents, either for monotherapy or as multi-drug therapies as drug-resistant strains are emerging worldwide, in particular mefloquine and chloroquine resistant strains. Therefore, new agents able to kill the resistant malaria parasites are warranted.
These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.
General Description
The solution now disclosed relates to antimalarial agents and to methods to obtain them. The advantage of the present disclosure is that there is no strain of P. falciparum known to be resistant to halogenated alkyl-aromatic secondary metabolite, in particular hierridin C and its derivatives. This advantage is relevant as death by malaria is almost exclusively caused by P. falciparum. 
The present disclosure relates to an antimalarial agent, in particular hierridin C, a halogenated alkyl-aromatic secondary metabolite, obtainable from the cyanobacterium Cyanobium sp. LEGE 06113, in particular the cyanobacterium Cyanobium sp. LEGE 06113 which was received on 29 May 2015 and accepted for deposit for patent purposes on 15 Jun. 2015 at the Scottish Association for Marine Science Culture Collection of Algae and Protozoa (CCAP), Oban, Argyll PA37 1QA, Scotland, United Kingdom—International Depositary Authority under the Budapest Treaty—under the CCAP number 1436/1.
Furthermore, the solution herein presented relates to processes used to obtain this compound from the cyanobacterium culture; furthermore it also relates to compounds sharing the same halogenated skeleton as hierridin C, and to its use in formulations for treating, preventing or inhibiting malaria in humans.
The hierridin C is a new antimalarial agent, which can be used to treat malaria, one of the most prevalent and life threatening infectious diseases in the developing world. It is a natural product and can be obtained from a renewable, photoautotrophic source by mass culturing in the laboratory or open ponds or alternatively it may be chemically synthetized.
Cyanobacteria are a large and widely spread group of photoautotrophic prokaryotes with a noteworthy ecological relevance being the only prokaryotes capable to carry out oxygenic photosynthesis, in which light energy is converted into chemical energy.
Cyanobacterial secondary metabolites represent a vast diversity of structures and have been isolated from a number of cyanobacterial genera, in particular cyanobacterial secondary metabolites have been mainly isolated from filamentous forms of the Oscillatoriales and Nostocales orders. Picocyanobacteria from the chroococcalean genera Cyanobium, Prochlorococcus and Synechococcus currently constitute the source of a very small fraction (less than 1%) of the reported cyanobacterial secondary metabolites. These cyanobacterial secondary metabolites may have potential therapeutic use.
Based on the International Union of Pure and Applied Chemistry (IUPAC) definitions, an alkyl group is defined as a univalent group derived from alkanes by removal of a hydrogen atom from any carbon atom —CnH2n+1. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H(CH2)n. The groups RCH2, R2CH(R≠H), and R3C(R≠H) are primary, secondary and tertiary alkyl groups, respectively. An aryl group is derived from arenes (monocyclic and polycyclic aromatic hydrocarbons) by removal of a hydrogen atom from a ring carbon atom.
“Alkyl” includes “lower alkyl” and extends to cover carbon fragments having up to 30 carbon atoms. Examples of alkyl groups include octyl, nonyl, norbornyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl-4-propylnonyl, 2-(cyclododecyl)ethyl, adamantyl, and the like.
“Lower alkyl” means alkyl groups of from 1 to 7 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2-methylcyclopropyl, cyclopropylmethyl, and the like.
Halogen is an element selected from the list consisting of: F, Cl, Br, I, At.
The half maximal inhibitory concentration (IC50) value indicates the concentration of a particular drug or other substance (inhibitor) necessary to inhibit a specific biological, biochemical or biological function or process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) function by half.
Antimalarial activity can be defined as the capability of compounds to inhibit the growth/viability of parasites. The half maximal inhibitory concentration (IC50) value for an antimarial drug indicates the concentration necessary to inhibit 50% of the parasites. The IC50s of hierridin C against the two parasite strains 3D7 (sensitive to chloroquine) and Dd2 (resistant to chloroquine and mefloquine) was in the low M range, which is identical to that of primaquine (an antimalarial in use despite its toxicity). Resistance indices (RI) of approximately 1 (RI=0.93) indicate that hierridin C kill the parasites via a different mechanism from chloroquine and mefloquine.
Hierridin B is a cyanobacterial secondary metabolite and the isolation of hierridin B from one of such strains, Cyanobium sp. LEGE 06113 was reported (Leão, P. N. et al. (2013) Antitumor Activity of Hierridin B, a Cyanobacterial Secondary Metabolite Found in both Filamentous and Unicellular Marine Strains. PloS ONE 8, doi:10.1371/journal.pone.0069562). This alkyl-aromatic metabolite had been previously isolated from a filamentous cyanobacterium, together with an extended chain analogue (Papendorf, O., König, G. M. & Wright, A. D. (1998) Hierridin B and 2,4-dimethoxy-6-heptadecyl-phenol, secondary metabolites from the cyanobacterium Phormidium ectocarpi with antiplasmodial activity. Phytochemistry 49, 2383-2386).
The polyhydroxylated aromatic moiety in hierridin B is hypothesized to originate from the action of a type III PKS, along the lines of the logic put forward for the cylindrocyclophanes (Nakamura, H. et al, 2012, Cylindrocyclophane biosynthesis involves functionalization of an unactivated carbon center. J Am Chem Soc 134(45):18518-21). Compound hierridin B showed modest, selective cytotoxicity towards HT-29 colon adenocarcinoma human cells (Leão, P. N. et al. (2013) Antitumor Activity of Hierridin B, a Cyanobacterial Secondary Metabolite Found in both Filamentous and Unicellular Marine Strains. PloS ONE 8, doi:10.1371/journal.pone.0069562).
An antiplasmodial activity was observed for a mixture of hierridin B and an extended-chain analogue (Papendorf, O., König, G. M. & Wright, A. D. (1998) Hierridin B and 2,4-dimethoxy-6-heptadecyl-phenol, secondary metabolites from the cyanobacterium Phormidium ectocarpi with antiplasmodial activity Phytochemistry 49, 2383-2386).
The low recovery (0.06%) of this metabolite (hierridin B) from the biomass of Cyanobium sp. LEGE 06113 hindered further evaluation of its bioactivity profile, and strongly motivated a re-isolation effort. While performing this task, another secondary metabolite was isolated from this cyanobacterium, hierridin C, which is a halogenated alkyl-aromatic secondary metabolite, in particular a chloro alkyl-aromatic secondary metabolite.
The present disclosure describes a new antimalarial agent isolated from laboratory cultures of the cyanobacterium Cyanobium sp. LEGE 06113, which has antimalarial activity against the strains of P. falciparum 3D7 and Dd2 (multidrug-resistant strain).
The present disclosure describes a compound of the formula I:
wherein:                R1 is a halogen;        R2, R4 are independently selected from a group consisting of C1-C6 alkyl, H, COCH3, COH, CO-alkyl, CO-aryl;        R3 is a C3-C30 alkyl;        or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof, for use in medicine (see FIG. 5).        
In an embodiment, the compounds previously described may comprise an R3 wherein the R3 is a C10-C30 alkyl.
In an embodiment, the compounds previously described may comprise R1, R2, R3 and R4 wherein                R1 may be selected from the group consisting of F, Cl, Br;        R2, R4 may be independently selected from a group consisting of C1-C6 alkyl;        R3 may be a C15-C21 alkyl.        
In an embodiment, R2 and R4 may be equal.
In an embodiment, the compound may be 3-chloro-4,6-dimethoxy-2-pentadecylphenol (see FIG. 4).
In an embodiment, the compounds previously described may be used for the treatment of malarian diseases.
The present disclosure also relates to a pharmaceutical composition comprising a compound as previously defined in any of the previous embodiments and a pharmaceutically acceptable carrier, adjuvant, excipient or mixtures thereof.
The present disclosure also relates to a pharmaceutical composition wherein it is administrated via topical, oral or parental or injectable.
The present disclosure also relates to a vaccine comprising a compound as previously described in any of the embodiments.
The present disclosure also relates to a process for obtaining compound 3-chloro-4,6-dimethoxy-2-pentadecylphenol comprising in the isolation of said compound from a solution comprising a cyanobacterium, in particular the cyanobacterium Cyanobium sp. LEGE 06113, which was received on 29 May 2015 and accepted for deposit for patent purposes on 15 Jun. 2015 at the Scottish Association for Marine Science Culture Collection of Algae and Protozoa (CCAP)—International Depositary Authority under the Budapest Treaty—under the CCAP number 1436/1.
The compound of formula I and its derivates, in particular, hierridin C and its derivates may also be used for the treatment of antimicrobian infections, namely as antimicrobial agent, in particular for the treatment or prevention of malarian diseases.
The compound/compositions can be combined with other excipients or active substances used in the context of veterinarian or human medicine.
The compound or compositions of the present disclosure can be administered by various routes, including topical, enteral and parenteral. Parenteral administration routes include intra-arterial, intra-articular, intracavitary, intradermal, intralympathic, intramuscular, intrasynovial, intravenous, or subcutaneous. Enteral routes include oral and gastro-intestinal. Topical routes include application into the skin and mucous membranes.
In a preferred embodiment, the composition is delivered to a patient by oral administration, which can be repeated according to a clinical prescription regime.
Dosage of the composition can be adapted to the administration route, as well as to the patient profile, including age, gender, condition, disease progression, or any other phenotypic or environmental parameters.
The composition may be in a solid form such as an amorphous, crystalline or semi-crystalline powder, granules, flakes, pills, capsules and suppositories. Such a solid form can be converted into a liquid form by mixing the solid with a physiologically appropriate liquid such as solvents, solutions, suspensions and emulsions.
In another aspect, the present invention provides a method of treating a patient with a microorganism infection disease, the method comprising administering an effective amount of compound or composition of the present disclosure to the patient.
In a further aspect, the present invention provides the compound or composition of the present disclosure for use in the treatment of microorganism infections, in particular malarian disease.
Further, the present invention provides the use of compound or composition of the present disclosure in the manufacture of a medicament for the treatment of microorganism infections, namely malaria disease.
In a particular aspect, the invention provides the composition described above for use in therapy. Further, the present invention provides the composition described above for use in the treatment/prevention of microorganism infections, namely malaria disease.
Throughout the description and claims the word “comprise” and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.